Presently, fluorescence polarization (anisotropy) immunoassays, which are based on the polarization or anisotropy of emitted light when a sample is excited with vertically polarized light, are widely used in clinical chemistry but are limited to the analysis of low molecular weight antigens such as drugs. This limitation exists because the short lifetime of the fluorescent probes preclude their use with larger, high molecular weight antigens which rotate more slowly in solution than do the smaller antigens.
With respect to metal-ligand complexes, there have been many reports which attempt to determine whether the excited state is distributed among the organic ligands on a rather symmetric complex, or whether it is localized between the metal and one of the ligands. This is an important distinction, because the former model predicts a low anisotropy, while the latter model predicts a higher anisotropy. An expert in the field would predict a low anisotropy, as symmetric molecules typically display low anisotropies, and metal ions in solution typically display zero anisotropies. Given the possibility of different effects which could account for loss of anisotropy, it was not clear that complexes such as Ru-metal-ligand complexes (Ru-MLC) would display useful anisotropy values. Also, even if the anisotropies were non-zero, it was not clear whether complexes such as Ru-MLC would display anisotropies which depend on molecular size, as needed for a fluorescence polarization immunoassay (FPI), or whether they would become depolarized by transfer of the excited state energy among the ligands, and thus would be independent of molecular size or rotational diffusion. Therefore, in regard to metal-ligand complexes, there has been no recognition of their use as fluorescent probes for biomedical applications.
The following references represent the state of the art of fluorescent polarization immunoassay:
Measurement of Angiotensinogen in Human Serum By Fluorescence Polarization Immunoassay, David B. Gordon, Clin. & Exper. Hyper.--Theory and Practice, A10(3), 1988 , pages 485-503. PA1 Immunoassay--Innovations in Label Technology, Joan H. Howanitz, M. D., Arch. Pathol. Lab. Med., Volume 112, August 1988, pages 775-779. PA1 Four Fluorescent Polarization Immunoassays for Therapeutic Drug Monitoring Evaluated, Virginia M. Havre et al, Clinical Chemistry, Volume 35, No. 1, 1989, pages 138-140. PA1 New Fluorescent Derivatives of Cyclosporin for Use in Immunoassays, M. T. French et al, Journal of Pharmaceutical & Biomedical Analysis, Volume 10, No. 1, 1992, pages 23-30. PA1 A Decade of Development of Immunoassay Methodology, James P. Gosling, Clinical Chemistry, Volume 36, No. 8, 1990, pages 1408-1427. PA1 Fluoroimmunoassay: Present Status and Key Problems, Erkki Soini et al, Clinical Chemistry, Volume 25, No. 3, 1979, pages 353-361. PA1 Fluorescent Excitation Transfer Immunoassay, Edwin F. Ullman et al, The Journal of Biological Chemistry, Volume 251, No. 14, Jul. 25, 1976, pages 4172-4178. PA1 Fluorescence Polarization in Immunochemistry, W. B. Dandliker et al, Immunochemistry, Volume 7, 1970, pages 799-828. PA1 Photophysics of Ruthenium Complexes Bound to Double Helical DNA, Challa V. Kumar et al, Journal American Chemical Society, Volume 107, No. 19, 1985, pages 5518-5523. PA1 Time-Resolved Fluorometry in Immunoassay, T. Lovgren et al, Alternative Immunoassays, 1985, pages 203-217. PA1 Current Concepts and Future Developments, R. P. Ekins, Alternative Immunoassays, 1985, pages 219-237. PA1 Immunoassays with Time-Resolved Fluorescence Spectroscopy: principles and Applications, Eleftherios P. Diamandis, Clinical Biochemistry, Volume 21, June 1988, pages 139-150. PA1 Europium Chelate Labels in Time-Resolved Fluorescence Immunoassays and DNA Hybridization Assays, Eleftherios P. Diamandis et al, Analytical Chemistry, Volume 62, No. 22, Nov. 15, 1990, pages 1149-1157. PA1 Europium as a Label in Time-Resolved Immunofluorometric Assays, Ilkka Hemmila et al, Analytical Biochemistry, 137 (1984), pages 335-343. PA1 Phosphorescent Immunoassay, Are Metalloporphyrins an Alternative to Rare Earth Fluorescent Labels?, A. P. Savitskii et al, Doklady Akademii Nauk SSSR, 1989, pages 48-51. PA1 Fiber-Optic Time-Resolved Fluorimetry for Immunoassays, Randy D. Petrea et al, Talanta, Volume 35, No. 2, 1988, pages 139-144. PA1 Applications of Lanthanide Chelates for Time-Resolved Fluoroimmunoassay, Philip Mottram et al, American Chemical Laboratory, May/June 1990, pages 34-38. PA1 U.S. Pat. No. 4,374,120 Feb. 15, 1983 Soini et al PA1 U.S. Pat. No. 4,745,076 May 17, 1988 Muller et al PA1 Design and Applications of Highly Luminescent Transition Metal Complexes, J. N. Demas et al, Analytical Chemistry, Volume 63, No. 17, Sep. 1, 1991, pages 829-837. PA1 Novel Fluorescent Label for Time-Resolved Fluorescence Immunoassay, Richard B. Thompson et al, SPIE, Volume 909, Time-Resolved Laser Spectroscopy in Biochemistry, 1988, pages 426-433. PA1 Redox Properties of Ruthenium(II) Tris Chelate Complexes Containing the Ligands 2,2'-Bipyrazine, 2,2'-Bipyridine, and 2,2'-Bipyrimidine, D. Paul Rillema et al, Inorganic Chemistry, Volume 22, No. 11, 1983, pages 1617-1622. PA1 Localization of Electronic Excitation Energy In Ru(2,2'-Bipyridine).sub.2 (2,2'-Bipyridine-4,4'-Dicarboxylic Acid).sup.2+ and Related Complexes, James Ferguson et al, Chemical Physics Letters, Volume 68, No. 1, pages 21-24. PA1 Energy Transfer from Luminescent Transition Metal Complexes to Oxygen, J. N. Demas et al, Journal of the American Chemical Society, May 25, 1977, pages 3547-3551. PA1 U.S. Pat. No. 4,565,790 Jan. 21, 1986 Hemmila et al PA1 U.S. Pat. No. 4,837,169 Jun. 6, 1989 Toner PA1 U.S. Pat. No. 4,962,045 Oct. 9, 1990 Picozza et al PA1 U.S. Pat. No. 5,089,423 Feb. 18, 1992 Diamandis et al PA1 U.S. Pat. No. 5,202,270 Apr. 13, 1993 Ungemach et al PA1 U.S. Pat. No. 5,221,605 Jun. 22, 1993 Bard et al PA1 U.S. Pat. No. 5,221,611 Jun. 22, 1993 Stenglein et al PA1 U.S. Pat. No. 5,061,857 Oct. 29, 1991 Thompson et al PA1 U.S. Pat. No. 5,083,852 Jan. 28, 1992 Thompson PA1 U.S. Pat. No. 5,094,819 Mar. 10, 1992 Yager et al PA1 Thin Layers of Depolarizers and Sensitizers, Lasovsky et al, Chem. Abstract 106, 1987: 95354n. PA1 Time-Resolved Photoselection of [Ru(bpy).sub.3 ].sup.2+ -exciton Hopping in the Excited State, Myrick et al, J. Amer. Chem. Soc. 109, 1987: 2841-2842. PA1 Circularly polarized Luminescence of Tris-Bipyridine Ruthenium (II) Complexes at Low Temperature, Tsubomura et al, Chem. Abstract 112, 1990: 65776h. PA1 coupling a luminescent asymmetric metal-ligand complex to the sample of interest to form a coupled sample; PA1 exciting the coupled sample with linearly polarized electromagnetic energy to cause the coupled sample to emit partially polarized fluorescent light; and PA1 measuring the polarization of the fluorescent light emission as a measure of a biological characteristic of the sample of interest. PA1 (a) mixing (1) an asymmetric metal-ligand complex conjugated to a molecule which specifically binds the analyte with (2) the sample; PA1 (b) exciting the mixture of step (a) with linearly polarized light to cause the complex to emit polarized light; PA1 (c) measuring the polarization of the light emitted by the complex; PA1 (d) calculating the amount of analyte in the sample by correlating the polarization measured in step (c) with the polarization of light emitted from a control sample containing a known amount of analyte. PA1 (a) mixing (1) a control containing a known amount of analyte conjugated to an asymmetric metal-ligand complex with (2) a molecule which specifically binds the analyte; PA1 (b) exciting the mixture of step (a) with linearly polarized light to cause the complex to emit polarized light; PA1 (c) measuring the polarization of the light emitted by the complex; PA1 (d) adding the sample to the mixture to form a new mixture including analyte not conjugated which competes with the analyte conjugated to the asymmetric metal-ligand complex in binding to the molecule which specifically binds the analyte, thereby causing a change in polarization; PA1 (e) measuring the change in polarization; PA1 (f) calculating the amount of analyte in the sample by correlating the change in polarization with the control containing a known amount of analyte. PA1 (a) mixing (1) a control containing a known amount of analyte conjugated to an asymmetric metal-ligand complex with (2) a molecule which has affinity for the analyte; PA1 (b) exciting the mixture of step (a) with linearly polarized light to cause the complex to emit polarized light; PA1 (c) measuring the polarization of the light emitted by the complex; PA1 (d) adding the sample to the mixture to form a new mixture including analyte not conjugated which competes with the analyte conjugated to the asymmetric metal-ligand complex in associating with the molecule which has affinity for the analyte, thereby causing a change in polarization; PA1 (e) measuring the change in polarization; PA1 (f) calculating the amount of analyte in the sample by correlating the change in polarization with the control containing a known amount of analyte.
The following references represent the state of the art of time-resolved immunoassay:
The following references disclose the known spectral properties of transition metal-ligand complexes:
The following references are of further background interest with respect to the present invention:
The present inventors wish to stress that none of the experts listed above have suggested complexes such as Ru or Os metal-ligand complexes for use in fluorescence polarization immunoassays. We refer to the emission from these complexes as fluorescence, primarily for convenience. The exact nature of the excited state is unknown, and the emission may be regarded as fluorescence or phosphorescence.
As indicated above, FPIs are presently limited to low molecular weight analytes, such as drugs and hormones, as admitted in Urios et al, "Adaptation of Fluorescence Polarization Immunoassay to the Assay of Macromolecules", Analytical Biochemistry, 185, 308-312 (1990) (which used F.sub.ab fragments having a molecular weight near 40,000, as opposed to a full IgG (Immunoglobin G, human) molecule having a molecular weight near 160,000) and Tsuruoka et al, "Fluorescence Polarization Immunoassay Employing Immobilized Antibody", Biosensors & Bioelectronics, 6, 501-505 (1991) (which bound the Ab to colloidal gold to increase its molecular weight in an attempt to change the correlation time of the larger antigens). With respect to Tsuruoka in particular, the present inventors do not consider its approach to be useful, since the molecular weight of the antibody is already too high for the lifetime of the label. In this regard, it is noted that the lifetimes of the probes are near 4 ns.
The limitation to low molecular weight analytes arises because the FPIs depend on a change in the apparent molecular weight of a fluorescently-labeled antigen upon binding to the antibody. A change in "apparent molecular weight" results upon binding to the large antibody molecule because the smaller antigen is now bound to the larger antibody molecule. Typical molecular weights of antigen and antibody are 1,000 and 160,000 daltons, respectively. The limitation to low molecular weight antigens is due to the short lifetimes of probes used in present FPIs and can be circumvented by using longer lifetime fluorophores. However, few such long-lived probes are known. The review articles mention pyrene derivatives, which display lifetimes near 100 ns. However, pyrene requires UV excitation near 320 nm, is photosensitive, and displays a low polarization. UV excitation results in significant autoflourescence from biological samples. A further advantage of the RuMLCs is their high chemical and photochemical stability.
To assist in understanding the above-described limitation and the present invention, some examples are set forth below. The need for a change in apparent molecular weight can be seen from the following example calculation. Suppose the labeled antigen has a molecular weight of 1,000 daltons, which results in a rotational correlation time of about 0.5 ns. The molecular weight of the antibody IgG is 160,000, resulting in a rotational correlation time near 100 ns (v+h.apprxeq.1.5, see eq. 8 below). The anisotropy of a fluorophore or labeled macromolecule is given by ##EQU1## where r.sub.0 is a constant typically near 0.3, .tau. is the lifetime and .theta. is the rotational correlation time.
For present immunoassays, the lifetimes of the probes are near 4 ns. The anisotropy of the free and antibody-bound antigens are thus as follows: ##EQU2## Hence, a large change in anisotropy is found upon binding of Ag to Ab for low molecular weight antigens.
The favorable change described above is not obtained for high molecular weight antigens. Suppose the molecular weight of the antigen is about 160,000, with a correlation time of 100 ns, and the molecular weight of the antibody is about 450,000, with a correlation time of 300 ns. The correlation time of the antigen-antibody complex will be near 400 ns. For the presently-used short lifetime fluorophores, the anisotropy values will be as follows: ##EQU3## This change in anisotropy is small because the lifetime is much shorter than the correlation time of the antigen.
The present invention provides a long lifetime label with good r.sub.0 values. In particular, data show lifetimes near 400 ns. For the larger molecular weight Ag-Ab complex ((.theta.=400 ns), the expected anisotropy values are as follows: ##EQU4##
The change in anisotropy for the 400 ns lifetime is 150%, which is much improved as compared to only 3% for the 4 ns lifetime. Also, many antigens of interest (e.g., IgM) are still larger (MW=950,000, .theta..gtoreq.600 ns), which will yield still higher anisotropy (r=0.180, % change=200%). It should be noted that the % change in anisotropy is more important than the absolute values.
In order to avoid confusion, it should be noted that polarization (P) and anisotropy (r) describe the same phenomena, and are related as follows: ##EQU5## where I.sub..parallel. and I.sub..perp. are the vertically and horizontally polarized components of the emission, when excited with vertically polarized light. The polarization and anisotropy are related by ##EQU6## The parameters P and r are both in common use. The values of P are used more often in FPI because they are entrenched by tradition and are slightly larger than the anisotropy values. The parameter r is preferred on the base of theory. Both P and r are related to the correlation time and/or molecular volume as follows: ##EQU7## In these equations k is the Boltzmann constant, T is the temperature (K), .eta. is the viscosity and V is the molecular volume. The correlation time is related to the molecular weight (M) of the protein as follows: ##EQU8## where R is the ideal gas constant, v is the specific volume of the protein and h is the hydration, typically 0.2 g H.sub.2 O per gram of protein.